Gene Expression
Nebulas
Gene Expression NebulasSummary: Natural Mucosal Barriers and COVID-19 in Children
Overall Design: BACKGROUND. Coronavirus disease 2019 (COVID-19) is more benign in children compared with adults for unknown reasons. This contrasts with other respiratory viruses where disease manifestations are often more severe in children. We hypothesize that a more robust early innate immune response to SARS coronavirus 2 (SARS-CoV-2) protects against severe disease. METHODS. Clinical outcomes, SARS-CoV-2 viral copies, and cellular gene expression were compared in nasopharyngeal swabs obtained at the time of presentation to the emergency department from 12 children and 27 adults using bulk RNA sequencing and quantitative reverse-transcription PCR. Total protein, cytokines, and anti–SARS-CoV-2 IgG and IgA were quantified in nasal fluid. We used a subset of 21 samples for RNAseq analysis. RESULTS. SARS-CoV-2 copies, angiotensin-converting enzyme 2 (ACE2), and TMPRSS2 gene expression were similar in children and adults, but children displayed higher expression of genes associated with IFN signaling, NLRP3 inflammasome, and other innate pathways. Higher levels of IFN-α2, IFN-γ, IP-10, IL-8, and IL-1β protein were detected in nasal fluid in children versus adults. Children also expressed higher levels of genes associated with immune cells, whereas expression of those associated with epithelial cells did not differ in children versus adults. Anti–SARS-CoV-2 IgA and IgG were detected at similar levels in nasal fluid from both groups. None of the children required supplemental oxygen, whereas 7 adults did (P = 0.03); 4 adults died. CONCLUSION. These findings provide direct evidence of a more vigorous early mucosal immune response in children compared with adults and suggest that this contributes to favorable clinical outcomes.
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| Treatment Protocol: | The swab was transferred to a tube containing media supplemented with 13 μM dithiothreitol to inactivate virus and then incubated in a thermomixer for 10 minutes at 37°C and 500 RPM. The cells were washed and frozen in 500 μL 90% FBS/10% DMSO. |
| Extract Protocol: | RNA was isolated from cryopreserved cells using the miRNeasy Micro kit (QIAGEN, 217084). Samples with sufficient RNA quantity and quality were used for analysis. Libraries were prepared at the Yale Center for Genome Analysis using the NEBNext rRNA Depletion kit (E6310L). Individual samples or pools from 2 patients of similar age and outcome (n = 4 pools, adults) were normalized to 1.2 nM and loaded on an Illumina NovaSeq S4 flow cell to generate 30 M read-pairs per sample. |
| Library Construction Protocol: | RNA libraries were prepared for sequencing in accordance with standard Illumina library preparation protocols. |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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